Structural basis for allosteric regulation of RyR1

项目摘要/摘要： Ryanodine受体介导的钙释放在肌肉收缩中起重要作用，并破坏 RyR介导的钙泄漏引起的可兴奋组织的钙稳态 疾病，以及随着年龄增长而导致的肌肉功能的逐渐丧失。RYR是 四聚体离子通道的大小不同寻常，每个亚基都有一个大的细胞质区域，充当 结合变构调节剂的支架，它通过结合远离 跨膜孔。RyR1活性的长程变构调节的机制基础不是很好 明白了。了解变构调节剂如何调节RyR1将有助于我们对RyR的理解 在生理环境中，并促进靶向通道的分子的发展以治疗RyR1 相关疾病。我们建议的目标是了解RyR1变构的结构作用机制 调制器使用单粒子低温电子显微镜(CRYO-EM)，辅之以功能分析。 我们将研究钙调蛋白和丹曲林作为长程变构调节剂的例子，这些变构调节剂 内源性蛋白质结合伙伴和小分子配体分别。我们将从三个方面着手实现这一目标 方向。在目标1中，我们寻求获得RyR1的完整原子模型，包括外围结构域 其中变构调节剂结合，这在很大程度上由于它们的灵活性和 机动性。我们将应用对称展开和掩码细化的组合来生成组合的 利用改进的周边区域局部分辨率进行重建，以及探索样本的优化 以获得更均匀的粒子集的准备。在目标2中，我们将获得RyR1的多个 钙调素和载脂蛋白复合体的功能状态，并使用突变体的单通道记录 钙调素和RyR1的结合，以检验由这些结构产生的假说。在目标3中，我们将调查 丹曲烯对RyR1的变构抑制机制--首次结构鉴定丹曲烯结合 然后研究丹曲林抑制和钙调蛋白调节之间的相互依赖关系， 镁和三磷酸腺苷。我们的研究将通过解开变构的结构基础而广泛地影响该领域 调节一个重要的离子通道，RyR1。丹曲烯结合部位的结构特征可能导致 肿瘤靶向治疗新药物RyR1的构效设计 体温过高与RyR1相关的肌病。

PROJECT SUMMARY/ABSTRACT: Ryanodine receptor-mediated calcium release plays an essential role in muscle contraction, and disruption of calcium homeostasis in excitable tissues caused by RyR-mediated calcium leak causes several genetic diseases, as well as contributing to the progressive loss of muscle function that occurs with aging. RyRs are tetrameric ion channels of unusually large size, with each subunit bearing a large cytosolic region that acts as a scaffold for the binding of allosteric modulators, which regulate the channel by binding at sites far from the transmembrane pore. The mechanistic basis of long-range allosteric modulation of RyR1 activity is not well understood. Understanding how allosteric regulators modulate RyR1 will both inform our understanding of RyR in a physiological context, and facilitate the development of molecules that target the channel to treat RyR1 related diseases. The goal of our proposal is to understand the structural mechanism of action of RyR1 allosteric modulators using single particle cryogenic electron microscopy (cryo-EM), complemented by functional analysis. We will investigate calmodulin and dantrolene as examples of long-range allosteric modulators which are endogenous protein binding partners and small molecule ligands, respectively. We will tackle this goal from three directions. In Aim 1, we seek to obtain a complete atomic model of RyR1, including of the peripheral domains where allosteric modulators bind, which have largely eluded sequence assignment due to their flexibility and mobility. We will apply a combination of symmetry expansion and masked refinement to generate a combined reconstruction with improved local resolution in peripheral regions, as well as explore optimization of sample preparation to obtain a more homogeneous particle set. In Aim 2, we will obtain structures of RyR1 in multiple functional states in complex with calmodulin and apocalmodulin, and to use single channel recordings of mutants of both calmodulin and RyR1 to test hypotheses arising from these structures. In Aim 3, we will investigate the mechanism of allosteric inhibition of RyR1 by dantrolene, by first structurally identifying the dantrolene binding site, and then examining the interdependence between inhibition by dantrolene and regulation by calmodulin, magnesium and ATP. Our research will broadly impact the field by unraveling the structural basis of allosteric regulation of an essential ion channel, RyR1. Structural characterization of the dantrolene binding site may lead the way to structure-based design of new RyR1 targeting therapeutics for the treatment of malignant hyperthermia and RyR1-related myopathies.